Lsa蛋白对金黄色葡萄球菌介导的炎性细胞因子表达的影响

doi:10.13560/j.cnki.biotech.bull.1985.2017-0731

生物技术通报 ›› 2018, Vol. 34 ›› Issue (1): 215-222.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0731

Lsa蛋白对金黄色葡萄球菌介导的炎性细胞因子表达的影响

卫瑞阳^1,2,3, 白杰^1,3, 徐彬^1,3, 席燕燕^1,3, 王琳燚^1,3, 王改利^1,3, 付趁^1,3, 魏凤仙^1,3, 李绍钰^1,3

1. 河南省农业科学院畜牧兽医研究所,郑州 450002;
2. 河南农业大学牧医工程学院,郑州 450002;
3. 河南省饲料与养殖环境控制工程技术研究中心河南省畜禽繁育与营养调控重点实验室,郑州 450002

收稿日期:2017-09-05 出版日期:2018-01-26 发布日期:2018-01-22
作者简介:卫瑞阳,男,硕士,研究方向：微生物与分子生物;E-mail：ruiyangwei@126.com;白杰为本文共同第一作者
基金资助:
国家现代农业产业技术体系项目（CARS-41）,国家重点研发计划项目（2016YFD0500509-09）,河南省农业科学院优秀青年基金项目（2016YQ20）

Effects of Lectin Subunit Alpha on the Expression of Staphylococcus aureus-stimulated Inflammatory Cytokines Mediated

WEI Rui-yang^1,2,3, BAI Jie^1,3, XU Bin^1,3, XI Yan-yan^1,3, WANG Lin-yi^1,3, WANG Gai-li^1,3, FU Chen^1,3, WEI Feng-xian^1,3, LI Shao-yu^1,3

1. Institute of Animal Husbandry and Veterinary Science,Henan Academy of Agricultural Sciences,Zhengzhou 450002;
2. College of Animal Science and Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002;
3. Henan Center for Feed and Aquaculture Environment Control Engineering Techniques Research,Henan Key Laboratory of Farm Animal Breeding and Nutritional Regulation,Zhengzhou 450002

Received:2017-09-05 Published:2018-01-26 Online:2018-01-22

摘要/Abstract

摘要： 试验旨在研究家蝇Lsa（Lectin subunit alpha）蛋白在金黄色葡萄球菌（Staphylococcus aureus）刺激下的炎性反应。通过慢病毒转染的方法,将构建好的载体pLEX-Lsa及pLEX分别转染到RAW264.7细胞中,通过筛选,获得稳定表达的RAW-pLEX-Lsa及RAW-pLEX细胞系,其中RAW-pLEX细胞作为阴性对照。然后通过S. aureus刺激细胞,收集未刺激和刺激后3 h,6 h,12 h和24 h的细胞,且收集未刺激和刺激6 h细胞上清。提取RNA,通过实时定量PCR（RT-PCR）检测炎性细胞因子TNF-a,IL-1β,NFκB-1 和NFκB-2基因的相对转录水平。通过酶联免疫吸附测定（ELISA）检测细胞上清中TNF-a和IL-1β的蛋白水平。RT-PCR结果显示,在S. aureus刺激后,与RAW-pLEX细胞相比,RAW-pLEX-Lsa细胞中TNF-a的两个转录剪接体的相对转录水平,TNF-α-tv-1在6 h显著下调（P<0.05）,TNF-α-tv-2在6 h和12 h显著下调（P<0.05）;RAW-pLEX-Lsa细胞中IL-1β的两个转录剪接体的相对转录水平,IL-1β-T在3 h,6 h和12 h显著下调（P<0.05）,IL-1β在3 h和6 h显著下调（P<0.05）;RAW-pLEX-Lsa细胞中NFκB-1 和NFκB-2基因的相对转录水平无明显下降趋势。ELISA检测结果显示,在S. aureus刺激6 h后,与RAW-pLEX细胞相比,RAW-pLEX-Lsa细胞上清中,TNF-a的蛋白水平显著下调（P<0.05）。结果表明,家蝇Lsa蛋白可以有效地发挥抗炎活性,显著地抑制TNF-α和IL-1β的转录和生产,而TNF-α和IL-1β的转录和生产的下调并不是通过抑制NFκB信号通路的活性引起的。

关键词: 凝集素, RAW264.7, 炎性细胞因子, NFκB, 转录剪接体

Abstract: The attempt of this work is to investigate the anti-inflammatory activity of Musca domestica lectin subunit alpha（Lsa）toward Staphylococcus aureus. The well-constructed pLEX-Lsa and pLEX were transfected into RAW264.7 cells with recombinant lentiviruses,then the stably-expressed RAW-pLEX-Lsa and RAW-pLEX were obtained after screening,and RAW-pLEX was as a negative control. S. aureus was used to stimulate RAW-pLEX-Lsa cells and RAW-pLEX cells,then both un-stimulated cells and stimulated cells for 3 h,6 h,12 h,and 24 h were collected,and the cell culture supernatants of 6 h un-stimulated and stimulated cells were also collected. Real-time quantitative reverse transcription PCR（RT-PCR）was performed to analyze the mRNA expression of TNF-a,IL-1β,NFκB-1 and NFκB-2,and the protein level of TNF-a and IL-1β in the supernatant were detected by ELISA. The results of RT-PCR showed that the stimulation of RAW-pLEX-Lsa by S. aureussignificantly down-regulated the mRNA expression of TNF-a transcript variant 1（TNF-a-tv-1）at 6 h（P<0.05）and TNF-a transcript variant 2（TNF-a-tv-2）at 6 h and 12 h（P < 0.05）,compared with the RAW-pLEX cells. The stable transfection of Lsa in RAW264.7 cells stimulated by S. aureus significantly down-regulated the mRNA expression of IL-1β-T at 3 h,6 h and 12 h（P < 0.05）and the mRNA expression of IL-1β at 3 h and 6 h（P < 0.05）,compared with the RAW-pLEX cells,while the mRNA expression of NFκB-1 and NFκB-2 in RAW-pLEX-Lsa had insignificant decreasing. The results of ELISA demonstrated that the protein level of TNF-a in the supernatants of RAW-pLEX-Lsa cells at 6 h after stimulation by S. aureus significantly down-regulated（P < 0.05）. In conclusion,Lsa possesses potent anti-inflammatory activity,and stable transfection of Lsa in RAW264.7 cells inhibits the expression and production of TNF-α and IL-1β. However,the inhibition of TNF-α and IL-1β is not resulted from blocking the activation of NF-κB signal pathway.

Key words: lectin, RAW264.7, inflammatory cytokines, NFκB, transcript variant

卫瑞阳, 白杰, 徐彬, 席燕燕, 王琳燚, 王改利, 付趁, 魏凤仙, 李绍钰. Lsa蛋白对金黄色葡萄球菌介导的炎性细胞因子表达的影响[J]. 生物技术通报, 2018, 34(1): 215-222.

WEI Rui-yang, BAI Jie, XU Bin, XI Yan-yan, WANG Lin-yi, WANG Gai-li, FU Chen, WEI Feng-xian, LI Shao-yu. Effects of Lectin Subunit Alpha on the Expression of Staphylococcus aureus-stimulated Inflammatory Cytokines Mediated[J]. Biotechnology Bulletin, 2018, 34(1): 215-222.

参考文献

[1] Peumans WJ, Van Damme EJ. Lectins as plant defense proteins[J]. Plant physiology, 1995, 109（2）：347-352.
[2] Barbosa PSF, Martins AMC, Toyama MH, et al. Purification and biological effects of a C-type lectin isolated from Bothrops moojeni[J]. Journal of Venomous Animals and Toxins including Tropical Diseases, 2010, 16（3）：493-504.
[3] Nunes ES, de Souza MA, Vaz AF, et al. Purification of a lectin with antibacterial activity from Bothrops leucurus snake venom[J]. Comparative Biochemistry & Physiology Part B Biochemistry & Molecular Biology, 2011, 159（1）：57-63.
[4] Soares GDSF, Assreuy AMS, Gadelha CADA, et al. Purification and biological activities of Abelmoschus esculentus, Seed Lectin[J]. The Protein Journal, 2012, 31（8）：674-680.
[5] Liu F, Tang T, Sun L, et al. Transcriptomic analysis of the housefly（Musca domestica）larva using massively parallel pyrosequencing[J]. Mol Biol Rep, 2012, 39（2）：1927-1934.
[6] Utarabhand P, Rittidach W, Paijit N. Bacterial agglutination by sialic acid-specific lectin in the hemolymph of the banana shrimp, Penaeus（Fenneropenaeus）merguiensis[J]. Sci Asia, 2007, 33：41-46.
[7] Zelensky AN, Gready JE. The C-type lectin-like domain superfamily[J]. Febs Journal, 2005, 272（24）：6179-6217.
[8] Drickamer K. C-type lectin-like domains[J]. Current Opinion in Structural Biology, 1999, 9（5）：585-590.
[9] Drickamer K. Evolution of Ca 2+ -dependent animal lectins[J]. Progress in Nucleic Acid Research and Molecular Biology, 1993, 45：207-232.
[10] Graham LM, Gupta V, Schafer G, et al. The C-type lectin receptor CLECSF8（CLEC4D）is expressed by myeloid cells and triggers cellular activation through Syk kinase[J]. J Biol Chem, 2012, 287（31）：25964-25974.
[11] Dambuza IM, Brown GD. C-type lectins in immunity：recent developments[J]. Current Opinion in Immunology, 2015, 32：21-27.
[12] Greenberg B. Flies and disease[J]. Scientific American, 1965, 213：92-99.
[13] Zhang Y, Wu S, Lv J, et al. Peste des petits ruminants virus exploits cellular autophagy machinery for replication[J]. Virology, 2013, 437（1）：28-38.
[14] Ogston A. On abscesses[J]. Review of Infectious Diseases, 1984, 6（1）：122-128.
[15] Uhlemann AC. Community-Associated Methicillin-Resistant Staphylococcus aureus Case Studies[J]. Methods in Molecular Biology, 2014, 1085：25-69.
[16] Ferrer M, Difrancesco LF, Liapikou A, et al. Polymicrobial intensive care unit-acquired pneumonia：prevalence, microbiology and outcome[J]. Critical Care, 2015, 19（1）：450
[17] Wardenburg JB. Panton-valentine leukocidin is not a virulence determinant in murine models of community-associated methicillin-resistant Staphylococcus aureus disease[J]. Journal of Infectious Diseases, 2008, 198（8）：1166-1170.
[18] Fast DJ, Schlievert PM, Nelson RD. Toxic shock syndrome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumor necrosis factor production[J]. Infection & Immunity, 1989, 57（1）：291-294.
[19] Gillet Y, Issartel B, Vanhems P, et al. Association between Staphylococcus aureus strains carrying gene for Panton-Valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients[J]. Lancet（London, England）, 2002, 359（9308）：753-759.
[20] Voyich JM, Otto M, Mathema B, et al. Is Panton-Valentine leukocidin the major virulence determinant in community-associated methicillin-resistant Staphylococcus aureus disease[J]? Journal of Infectious Diseases, 2006, 194（12）：1761-1770.
[21] Mogensen TH. Pathogen recognition and inflammatory signaling in innate immune defenses[J]. Clinical Microbiology Reviews, 2009, 22（2）：240-273.
[22] Nau R, Eiffert H. Modulation of release of proinflammatory bacterial compounds by antibacterials：potential impact on course of inflammation and outcome in sepsis and meningitis[J]. Clinical Microbiology Reviews, 2002, 15（1）：95-110.
[23] Mukhin AG, Ivanova SA, Knoblach SM, et al. New in vitro model of traumatic neuronal injury：evaluation of secondary injury and glutamate receptor-mediated neurotoxicity[J]. Journal of Neurotrauma, 1997, 14（9）：651-663.
[24] Aribi M, Meziane W, Habi S, et al. Macrophage bactericidal activities against Staphylococcus aureus are enhanced in vivo by selenium supplementation in a dose-dependent manner[J]. PLoS One, 2015, 10（9）：e0135515.
[25] Porcherie A, Cunha P, Trotereau A, et al. Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells[J]. Veterinary Research, 2012, 43（1）：14.
[26] Zhu F, Yue W, Wang Y. The nuclear factor kappa B（NF-κB）activation is required for phagocytosis of Staphylococcus aureus, by RAW 264. 7 cells[J]. Experimental Cell Res, 2014, 327 （2）：256-263.
[27] Larsen GL, Henson PM. Mediators of inflammation[J]. Annual Review of Immunology, 1983, 1（1）：335-359.
[28] Murdaca G, Spanò F, Contatore M, et al. Infection risk associated with anti-TNF-α agents：a review[J]. Expert Opinion on Drug Safety, 2015, 14（4）：1-12.
[29] Koca SS, Bahcecioglu IH, Poyrazoglu OK, et al. The Treatment with antibody of TNF-α reduces the inflammation, necrosis and fibrosis in the non-alcoholic steatohepatitis induced by methionine- and choline-deficient diet[J]. Inflammation, 2008, 31（2）：91-98.
[30] Miller LS, Pietras EM, Uricchio LH, et al. Inflammasome-mediated production of IL-1beta is required for neutrophil recruitment against Staphylococcus aureus in vivo[J]. Journal of Immunology, 2007, 179（10）：6933-6942.
[31] Pires AF, Rodrigues NVFC, Soares PMG, et al. A novel N-acetyl-glucosamine lectin of Lonchocarpus araripensis attenuates acute cellular inflammation in mice[J]. Inflammation Research, 2016, 65（1）：43-52.
[32] Shan M, Gentile M, Yeiser JR, et al. Mucus enhances gut homeostasis and oral tolerance by delivering immunoregulatory signals[J]. Science, 2013, 342（6157）：447-453.
[33] Freitas RS, do Val DR, Fernandes MEF, et al. Lectin from Abelmoschus esculentus reduces zymosan-induced temporomandibular joint inflammatory hypernociception in rats via heme oxygenase-1 pathway integrity and tnf-α and il-1β suppression[J]. International Immunopharmacology, 2016, 38：313-323.
[34] Jin H, Yang X, Liu K, et al. Effects of a novel peptide derived from human thrombomodulin on endotoxin-induced uveitis in vitro and in vivo[J]. FEBS letters, 2011, 585（21）：3457-3464.
[35] Matsumoto T, Takahashi N, Kojima T, et al. Soluble Siglec-9 suppresses arthritis in a collagen-induced arthritis mouse model and inhibits M1 activation of RAW264. 7 macrophages[J]. Arthritis Research & Therapy, 2016, 18（1）：133.

Lsa蛋白对金黄色葡萄球菌介导的炎性细胞因子表达的影响

Effects of Lectin Subunit Alpha on the Expression of Staphylococcus aureus-stimulated Inflammatory Cytokines Mediated

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	吴航, 李智强, 刘文德. 稻瘟病菌分泌蛋白MoCDIE2诱导植物细胞死亡的功能分析[J]. 生物技术通报, 2020, 36(1): 15-22.
[2]	林师,张丽莉,王国栋. 杂色鲍两种perlucin基因的克隆与表达分析[J]. 生物技术通报, 2016, 32(12): 113-123.
[3]	刘玎, 陈劲, 刘志, 朱生伟. 掌叶半夏凝集素基因PPA2抗蚜功能分析[J]. 生物技术通报, 2016, 32(10): 180-187.
[4]	蔡佳, 张雪利, 吴灶和, 简纪常, 鲁义善, 冯东岳, 焦茂兴. 红笛鲷（Lutjanus sanguineus）C型凝集素基因的克隆与组织表达分析[J]. 生物技术通报, 2015, 31(5): 179-185.
[5]	孟建宇;宋馨宇;. 蒙古口蘑子实体凝集素性质初步研究[J]. , 2012, 0(02): 188-192.
[6]	王艳玲;李东;王秀利;. 海参免疫相关基因的研究进展[J]. , 2011, 0(09): 22-26.
[7]	谢广成;李丹彤;周立敬;. 刺参甘露糖结合凝集素的生物信息学分析[J]. , 2011, 0(02): 136-140.
[8]	陈石;李春雨;郑加协;周红玲;颜元培;方志坚;易干军;. 香蕉凝集素BanLec基因转化毕赤酵母及转化子的鉴定[J]. , 2010, 0(06): 121-123.
[9]	王颖;仇雪梅;王娟;王秀利;. 刺参病害现状及其生物技术检测的研究进展[J]. , 2009, 0(11): 60-64.
[10]	张利鑫;刘强;. 添加N-乙酰-D-半乳糖胺消除大豆凝集素抗营养活性的可行性研究[J]. , 2009, 0(07): 156-159.
[11]	陈礼刚;陈晓艺;郭继强;刘志文;李宪臻;. 糖微阵列技术在糖组学研究中的进展[J]. , 2009, 0(06): 67-70.
[12]	程茂高;乔卿梅;原国辉. 植物外源抗虫基因及其应用[J]. , 2004, 0(05): 18-20.
[13]	汪开治. 利用4种血清标志物筛查唐氏综合征的新方法[J]. , 2003, 0(05): 42-42.
[14]	汪开治. 印度转基因食用作物现状[J]. , 2003, 0(03): 48-52.
[15]	高莹;瞿礼嘉;陈章良. 植物凝集素的分子生物学研究[J]. , 2000, 0(05): 18-22.